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Prevalence of Cannabinoid Use in Patients With Hip and Knee Osteoarthritis

From the Department of Orthopedics, Mayo Clinic Arizona, Phoenix, AZ (Dr. Deckey, Dr. Lara, Dr. Hassebrock, Dr. Spangehl, and Dr. Bingham), and the Department of Orthopedics, Loma Linda Medical Center, Loma Linda, CA (Dr. Gulbrandsen).

Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Orthopaedic Surgeons.

This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction:

State legalization and widespread marketing efforts have increased the accessibility and consumption of off-label, non–FDA-approved, cannabinoid (CBD) products. Although clinical evidence is largely absent for the treatment of musculoskeletal pain, patients are experimenting with these products in efforts to relieve joint pain. Assessment of the prevalence, perceived efficacy compared with other nonsurgical modalities, and usage patterns is warranted. The purpose of this study was to report the prevalence and perceived self-efficacy of CBD products in patients with symptomatic hip and/or knee osteoarthritis (OA).

Methods:

Two-hundred consecutive patients presenting with painful hip or knee OA were surveyed at their initial evaluation at a large academic center. Using Single Assessment Numeric Evaluation (SANE) scores, survey questions assessed perceived pain and effectiveness of CBD products, in addition to other nonsurgical treatment modalities. Chart review provided demographic factors. Descriptive statistics were used to characterize the data.

Results:

Of the 200 patients (80 hip OA, 108 knee OA, and 12 both), 66% were female, and average age was 67 years (range 36 to 89 years). Twenty-four percent (48/200) of patients endorsed use of CBD products before their presentation. The average presenting SANE score (range 0 to 100) for non-CBD users was 50.8 compared with 41.3 among CBD users (P = 0.012). Sixty percent of patients learned about CBD through friends, and 67% purchased CBD directly from a dispensary. Oral tinctures (43%) and topical applications (36%) were the most commonly used forms. In addition, 8% of participants in this study had tried marijuana for their pain.

Conclusion:

A 24% incidence of CBD usage was found among patients presenting with hip or knee OA. No significant perceived benefit of CBD use seems to exist compared with its nonuse, as patients who used CBD reported significantly worse SANE and visual analogue scale scores than nonusers at baseline. Follow-up studies are warranted to assess these findings.

State legalization and widespread marketing efforts have increased the accessibility and consumption of off-label, non–FDA-approved, cannabinoid (CBD) products. Subsequently, these products have been promoted for the treatment of numerous ailments, including joint pain. Although clinical evidence is largely absent for the treatment of musculoskeletal pain, patients are experimenting with these products in efforts to relieve joint pain. 1,2,3,4,5,6 If proven effective, these medications could provide multimodal pain control in the treatment of arthritis-related pain.

Surgeons should be aware of the effects of over-the-counter medications, especially non–FDA-approved medications that their patients are consuming. Given the increased availability of CBD products, investigations into the prevalence and perceived efficacy of CBD for treatment of osteoarthritis (OA) are warranted. To our knowledge, data evaluating the prevalence and perceived efficacy of CBD products for the treatment of OA are limited. Therefore, the purpose of this study was to report the prevalence and subjective efficacy of CBD products in patients with symptomatic hip and/or knee OA presenting for an initial orthopaedic surgery consultation.

Methods

After institutional review board approval, 200 consecutive patients presenting with painful hip or knee OA were surveyed at their initial arthroplasty clinic evaluation at a single high-volume academic center. As part of the initial intake screening, patients were asked to complete a 21-question survey. Questions concerning function and perceived efficacy of treatments were assessed using Single Assessment Numeric Evaluation (SANE) on a 1 to 100 point scale, with a score of 100 indicating the highest perceived benefit (SANE). 7,8 In addition, medical chart review was undertaken for background demographic factors.

After completion of questionnaires (see appendix for questionnaire example, Appendix 1, http://links.lww.com/JG9/A108), answers were categorized and tabulated. Average SANE scores for interventions were calculated as well. Questions results were binary (yes/no), numeric (SANE/visual analogue scale [VAS]), or free text (ex “Question 14: ‘How did you hear about CBD?’”). Free text answers were manually reviewed for each respondent and categorized into nominal reviewable outcomes (Table ​ (Table5). 5 ). Radiographs for every patient were reviewed by two independent reviewers. Descriptive statistics were performed to characterize the population; T-tests were used to compare the variation of continuous variables. Comparison of proportions for sample populations was performed with z-tests. All statistical analysis was performed with JMP statistical software (SAS Institute).

Table 5

Characterization of CBD Use and Procurement Among the Sample Population

Descriptor N
Referral source
HCP 7
Friend 31
Advertisement 13
Work 1
Purchasing location
HCP 1
Friend 3
Online 10
Store 29
CBD type
Capsule 5
Topical 16
Oil tincture 19
Edible 4
Frequency of use
Daily 13
Twice daily 6
Three times daily 2
As needed 19
Only once 4

CBD = cannabinoid, HCP = healthcare provider

Results

Of the 200 consecutive patients, 100% completed the survey. Sixty-six percent were female, and the average age was 67 years. Knee OA was the most common complaint (n = 108) followed by hip OA (n = 80), and a minority of patients had symptoms in both joints at presentation (n = 12). Thirty-seven percent of these patients were symptomatic on the right side, 31% on the left side, and 32% presented with bilateral complaints. Knee OA had an average Kellgren-Lawrence OA grade of 2.7 (range 0 to 4). Average Tönnis scale grading of the affected hip OA was 1.8 (range 0 to 3) (Table ​ (Table1 1 ).

Table 1

Demographic and Radiographic Variables of Arthroplasty Clinic Sample Population

No. of patients, n 200
Age (y) (±SD) 67.21
Female, n (%) 112 (56)
Joints, n (%)
Knee 108 (54)
Hip 80 (40)
Both 12 (6)
Laterality, n (%)
Left 62 (31)
Right 74 (37)
Both 64 (32)
Knee osteoarthritis grade a (n = 159), n (%)
2 (1.1)
1 29 (18.3)
2 34 (21.4)
3 42 (26.4)
4 52 (32.7)
Hip osteoarthritis grade b (n = 107), n (%)
7 (6.5)
1 36 (33.6)
2 31 (29.1)
3 33 (30.8)

Twenty-four percent (48/200) of patients endorsed use of CBD products before their presentation. The average presenting SANE score (range 0 to 100) for non-CBD users was 50.8 compared with 41.3 among CBD users (P = 0.012). The average VAS score (range 0 to 10) for non-CBD users was 5.7 compared with 6.6 among CBD users (P = 0.036). No difference in the asymptomatic contralateral joint SANE score (range 0 to 100) was found when comparing non-CBD users with CBD users (81.9 versus 75.9, respectively, P = 0.129) (Table ​ (Table2 2 ).

Table 2

SANE and VAS Scores Among Non-CBD and CBD Users, Respectively

Factor Non-CBD Users (n = 152), n (%) CBD Users (n = 48), n (%) P Value
Symptomatic joint SANE (average) 50.8 41.3 0.012
Contralateral unaffected joint SANE (average) 81.9 75.9 0.129
VAS pain rating (average) 5.7 6.6 0.036

CBD = cannabinoid, SANE = Single Assessment Numeric Evaluation, VAS = Visual Analogue Scale

Among non-CBD users, 73% had tried NSAIDs for symptomatic relief compared with 90% among the CBD using group. A statistically higher percentage of patients in the CBD group had used NSAIDs for symptomatic relief compared with non-CBD users (P = 0.017). No significant difference was found in the number of patients who had tried bracing treatment, steroid injections, or viscosupplementation injections between the two groups. A significantly higher percentage of marijuana use was found among the CBD group compared with non-CBD users (31% versus 1%, respectively, P < 0.001) despite similar rates of “Other” recreational drug use (15% CBD users versus 11% non-CBD users) (Table ​ (Table3 3 ).

Table 3

Frequency of Alternative Treatments for Symptomatic Osteoarthritis Used by Study Sample Population Non-Cannabinoid (CBD) and CBD Users, Respectively

Factor Non-CBD Users (n = 152), n (%) CBD Users (n = 48), n (%) P Value a
NSAID 111 (73) 43 (90) 0.017
Bracing treatment 43 (28) 26 (54) 0.289
Steroid injection 79 (52) 28 (58) 0.119
Viscosupplementation injection 30 (20) 11 (23) 0.575
Marijuana 2 (1) 15 (31)
Recreational “other” drug use 16 (11) 7 (15) 0.928

A significant difference was seen after NSAID use; non-CBD users reported an improvement with an increase in the average SANE to 52.7, whereas CBD users decreased to a SANE of 39.0 (P = 0.012). Otherwise, the differences in SANE scores between the two groups after bracing treatment, steroid injection, viscosupplementation injection, or marijuana use were not statistically significant (Table ​ (Table4 4 ).

Table 4

SANE Score Averages Among Two Groups After Nonsurgical Treatments

Average SANE Scores Non-CBD Users (n = 152), n CBD Users (n = 48), n P Value
Baseline 50.8 41.3 0.012
Post-NSAID 52.7 39.0 0.012
Post–bracing treatment 40.2 37.6 0.727
Post-steroid 54.9 45.9 0.205
Post-viscosupplementation 55.0 43.4 0.225
Post-marijuana 25.0 47.0 0.319

CBD = cannabinoid, SANE = Single Assessment Numeric Evaluation

Among CBD users, 60% of patients learned about CBD through friends, and 67% purchased CBD directly from a dispensary. Oral tinctures (43%) and topical applications (36%) were the most commonly used forms of CBD. Twenty-two percent of all the patients in this sample reported ongoing CBD utilization (Table ​ (Table5 5 ).

Discussion

In this prospective cohort of 200 consecutive patients, 24% (48 patients) reported trying CBD-containing products for relief of their arthritis-related pain before their initial orthopaedic surgical consultation. Although CBD use has not been previously characterized in this population, its prevalence is similar to the reported 15% to 22% of the general US population that reported marijuana use. 9,10 However, this reported CBD use is much higher compared with marijuana use in an older population. Han and Palamar 11 found that 9% of adults aged 50 to 64 years and 2.9% of adults aged 65 years and older reported marijuana use, which was similar to the 9% of patients who reported marijuana use in our study. This large difference in CBD and marijuana usage in a similarly aged population demonstrates the growing trend and popularity of CBD utilization. Given that more and more patients will arrive in clinic having tried or wanting to try these products, it is crucial that the orthopaedic surgeon is aware of CBD products and current trends in utilization. In addition, in the setting of the opioid crisis, it is imperative that we continue to identify new and potentially less-addictive modalities for pain relief. The goal of this study was to characterize and analyze CBD usage and perceived effectiveness in patients presenting for primary consultation with hip and/or knee OA.

To understand why CBD has become such a rapidly growing trend, a brief history is helpful. The passage of the US Hemp Farming Act of 2018 removed hemp (defined as cannabis with less than 0.3% tetrahydrocannabinol [THC]) from Schedule I Controlled Substances. 12 CBD can be derived from cannabis, which comes from the plant Cannabis sativa. Virtually overnight, a new US industry was created. This industry brought with it a legal, unregulated product with broad claims of treating anxiety, insomnia, PTSD, and reducing pain and inflammation. Although not containing high percentages of THC, hemp can still contain CBD, which augments the body’s endogenous CBD system primarily through CB1 and CB2 receptors in both the central and peripheral nervous system. These receptors have been shown to play roles in modulating nociception and inflammatory pathways. 13 However, the full effects of CBD are still not fully understood. Although animal models have shown CBD to decrease OA-related pain, 14,15,16,17,18,19 its efficacy in humans has not been fully supported. 18,20,21

As the stigma surrounding THC and CBD use decreases and these products become more readily available, the prevalence of their use will likely increase. Previously, research has been hampered by lack of funding and the Schedule I classification of cannabis. Given the wide availability of CBD in the United States at present and movements to remove cannabis from the Schedule I classification, it is believed that more knowledge about how THC/CBD functions will come to light. A study using National Inpatient Sample database showed that marijuana/THC use was associated with decreased mortality in patients undergoing total hip arthroplasty (THA), total knee arthroplasty (TKA), total shoulder arthroplasty (TSA), and traumatic femur fixation. 22 In addition, two previous, recently published studies in the orthopaedic literature have explored the use of CBD and THC in arthroplasty. 4,5 Hickernell et al 4 examined the use of dronabinol, a synthetic form of THC, in a multimodal pain regimen after THA and TKA surgery. In their study, the group taking a prescribed dose of drocannabinol had significantly shorter stays and significantly fewer total morphine equivalents. However, this was a small (81 patients) retrospective study and warrants further studies to fully support this trend. Runner et al 5 found that 16.4% of patients following TKA or THA reported use of CBD or THC in the perioperative period. Compared with nonusers, no significant difference was observed in the length of narcotic use, total morphine equivalents used, postoperative pain scores, or the length of stay. Patients in this study were self-medicating without uniformity, which is in contrast to the prescribed dose of drocannabinol used in the Hickernell study.

Our study, however, showed no significant perceived benefit of CBD use compared with nonuse, and patients who used CBD actually reported significantly worse SANE and VAS scores at baseline than nonusers. The symptomatic joint(s)’ SANE score significantly differed between CBD users and nonusers at initial presentation (41.3 versus 50.8, P = 0.012). Previous literature has suggested that the minimally clinically important difference for knee injury interventions is approximately 7 to 19, suggesting that perhaps baseline presentation SANE scores may have been statistically different but not clinically measurable. 23 In addition, VAS pain rating for CBD users was significantly higher at baseline than nonusers (6.6 versus 5.7, P = 0.036). Interestingly, patients who used CBD products were also significantly more likely to use NSAIDs. This finding suggests that the patients taking CBD products may have had more symptomatic OA or more prone to self-medicating. Patients who reported CBD use were also significantly more likely to report marijuana use.

Several limitations of this study must be acknowledged. Although this was a prospective study, recall bias may be present as patients were asked to recall use of treatment and its effectiveness leading up to their first visit. In addition, only patients presenting for primary hip and knee arthroplasty consultation were included in this study. This restriction limits the generalizability of our findings to other orthopeadic specialties. Future studies are warranted in other subspecialties, such as sports medicine, where injuries are more acute. The perceived efficacy of CBD products may be different for acute pain than for chronic pain. The source of CBD product and route of administration was also not standardized, which may play a role in its effectiveness. In addition, this study had a limited sample size of 200 patients and as such may be subject to type 2 error when concluding no difference. Therefore larger, multicenter studies are needed to fully evaluate CBD use in this population and to enhance generalizability as well as a randomized controlled trial with placebo and a controlled dose of CBD. Finally, a substratification of severity of OA in either group would be useful in future studies attempting to determine the efficacy of CBD in symptomatic relief.

Conclusion

To our knowledge, this is the first prospective study to evaluate the usage of over-the-counter CBD products in a hip and knee OA population. A 24% incidence of CBD usage was found among these patients. We found no significant perceived benefit of CBD use compared with nonuse, and patients who used CBD actually reported significantly worse SANE and VAS scores than nonusers.

Footnotes

None of the following authors or any immediate family member has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this article: Dr. Deckey, Dr. Lara, Dr. Gulbrandsen, Dr. Hassebrock, Dr. Spangehl, and Dr. Bingham.

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Cannabidiol (CBD): a killer for inflammatory rheumatoid arthritis synovial fibroblasts

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Associated Data

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abstract

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Introduction

Cannabidiol (CBD) is a non-intoxicating cannabinoid found in cannabis sativa 1 . In contrast to the psychoactive constituent tetrahydrocannabinol (THC), CBD demonstrates no direct effect at cannabinoid receptors 1 and 2 (CB1 and CB2) but modulates the effect of agonists suggesting an allosteric function 2 . In addition, CBD binds to PPARγ, GPR3/6/12/18/55, TRPV1/2, TRPA1, 5-hydroxytryptamine receptor, and mitochondrial proteins 3–11 . Despite its promiscuous pharmacology, CBD is well tolerated even when given in high concentrations 12,13 . Side effects of CBD in humans include diarrhea and fatigue and, more importantly, CBD interacts with other drugs since it is metabolized by CYP enzymes in the liver thereby inhibiting the degradation of other therapeutic compounds 14,15 . While the therapeutic benefits of CBD in childhood epilepsy are well documented, its effects on inflammation have only been investigated in animal models 13,16 . Studies in rodents with osteoarthritis or collagen-induced arthritis demonstrated anti-inflammatory and analgesic effects of CBD, but these studies did not identify the mechanism of action 17–19 . Here, we investigate the effect of CBD on intracellular calcium, cell viability, and cytokine production in rheumatoid arthritis synovial fibroblasts (RASF). RASF are one major contributor of joint destruction in RA as they secrete pro-inflammatory cytokines and matrix degrading enzymes 20 . In fact, subsets of RASF selectively mediate joint destruction or the inflammatory response, emphasizing their important role in the pathogenesis of RA 21 . In previous studies, we already identified TRPA1 as a therapeutic target since the TRPA1 agonist Polygodial selectively deleted TNF-activated RASF 22 . CBD also binds TRPA1 7,23 , and therefore we hypothesized that CBD has detrimental effects on cell viability, which might explain in part its mechanism of action at sites of inflammation.

Results

CBD reduces cell viability and proliferation of RASF

Over the course of 6 h we found that CBD (≥5 µM) decreases cell viability (Fig. 1a, b ), but a stimulatory effect was detected for 1 µM CBD in TNF pre-incubated RASF (Fig. ​ (Fig.1b). 1b ). CBD combined with the TRPA1 antagonist, A967079, recovered cell viability (Fig. ​ (Fig.1h). 1h ). TRPA1 is upregulated by TNF (Fig. ​ (Fig.1f) 1f ) and we also detected an increase of TRPA1 mRNA by real-time PCR after 24 h under the influence of TNF (data not shown). Ruthenium Red (RR) also reduced the detrimental effects of CBD (Fig. ​ (Fig.1i). 1i ). Surprisingly, 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), supported cell viability at low CBD concentrations but enhanced its cytotoxic effects at concentrations ≥1 µM (Fig. ​ (Fig.1j), 1j ), while Cyclosporin A (CsA), blocked the effects of CBD (Fig. ​ (Fig.1k). 1k ). Since RealTime-Glo assays were conducted at 37 °C in serum-free medium but without CO2 and humidity control, we also confirmed these results in cell titer blue endpoint assays (Fig. ​ (Fig.5d). 5d ). In vivo, CBD is bound to lipoproteins/albumin lowering the available concentration of free CBD 24 . Therefore, we investigated the effect of fetal calf serum content with CBD on proliferation. CBD in concentrations ≥5 µM reduced proliferation of RASF in medium without or 2% FCS (Fig. 1c, d ). TNF pre-stimulation enhanced proliferation at 5 µM CBD in 0% FCS (Fig. ​ (Fig.1c) 1c ) while the opposite was true using 2% FCS (Fig. ​ (Fig.1d). 1d ). With 10% FCS, CBD inhibited proliferation of RASF only at 20 µM (Fig. ​ (Fig.1e). 1e ). DMSO (vehicle control) alone had a stimulatory effect on proliferation (Fig. ​ (Fig.1e, 1e , green line). These findings underline the need for relatively high concentrations of CBD when used in in vivo settings 13 .

a, b Mean cell viability of unstimulated (a) or TNF pre-stimulated (b) RASF after CBD challenge monitored in real-time over the course of 375 min. ce Mean proliferation of RASF with (red bars) and without (white bars) TNF pre-stimulation in response to CBD in medium containing 0% FCS (c), 2% FCS (d), and 10% FCS. The dotted line represents the unstimulated control, which was set to 100%. f Flow cytometric detection of TRPA1 protein in RASF with or without TNF stimulation for 72 h. gk Mean cell viability of TNF pre-stimulated RASF after CBD challenge and concomitant addition of inhibitors over the course of 20 h. n is the number of replicates and patient samples investigated. ANOVA with Dunnett’s T3 post-hoc test was used for comparisons in a, b, gk. ANOVA with Bonferroni post-hoc test was used for comparisons in ce. Two-tailed t-test was used for comparisons in f. *p < 0.05; **p < 0.01, ***p < 0.001 vs control. The error bars in cf represent the standard error of mean (sem).

RASF were incubated for 72 h with TNF. After wash-off, RASF were challenged with antagonists for 30 min followed by CBD addition for 72 h. ANOVA was used for all comparisons vs control w/o CBD. a, c, e, g IL-6, IL-8, MMP-3 production, and cell number after 72 h challenge with CBD. b, d, f, h IL-6, IL-8, MMP-3 production, and cell number after 72 h challenge with CBD (10 µM and 20 µM) with concomitant addition of the TRPA1 antagonist A967079 and the mPTP inhibitor CsA. Significant differences between CBD in different concentrations are depicted as *p < 0.05, **p < 0.01, and ***p < 0.001, and CBD versus CBD/antagonist treatment are depicted as #p < 0.05, ##p < 0.01, and ###p < 0.001. ANOVA with Bonferroni post-hoc test was used for all comparisons. The dotted line in g, h represents the control value which was set to 100%. n is the number of replicates out of four different patient samples. The error bars represent the standard error of mean.

CBD increases intracellular calcium and its effects are enhanced by TNF

CBD influences calcium mobilization 9,23,25 and we found that concentrations ≥5 µM increased intracellular calcium (Fig. ​ (Fig.2a). 2a ). The potency of CBD was enhanced by pre-incubation with TNF for 72 h (Fig. ​ (Fig.2b). 2b ). Under these conditions, intracellular calcium levels were significantly increased compared to untreated RASF (Fig. ​ (Fig.2d). 2d ). When extracellular calcium was omitted by using PBS, CBD still increased intracellular calcium although to a smaller extent (Fig. ​ (Fig.2c) 2c ) Besides calcium, we also analyzed the uptake of the cell viability dye PoPo3 iodide under CBD stimulation. PoPo3 uptake increases when membrane integrity is compromised during apoptosis or necrosis. CBD dose-dependently increased the uptake of PoPo3 which was enhanced by extracellular calcium and TNF pre-stimulation (Fig. 2f, g ). Basal uptake of PoPo3 and intracellular calcium levels were increased by TNF pre-treatment (Supplementary Fig. 3A, B). The detrimental effect of CBD on cell viability was also confirmed in the XCELLigence system using untreated RASF (Supplementary Fig. 1).

Calcium mobilization and PoPo3 uptake partly depend on TRPA1 activation

Since CBD binds TRPV1, TRPV2, and TRPA1 7 we investigated the involvement of these ion channels. RR, a general inhibitor for several TRP channels 26–28 , reduced intracellular calcium levels (Fig. 3e, f ) and PoPo3 uptake (Fig. 3g, h ) but the magnitude of inhibiton was small. RR did not change basal calcium levels or PoPo3 uptake in unstimulated but did so in TNF pre-stimulated RASF (Supplementary Fig. 3). Next, we combined CBD with the TRPV1 antagonist Capsazepine (CPZ) 29 . CPZ had only a minor influence on CBD-induced calcium levels and PoPo3 uptake (Fig. 3i–l ). Of note, CPZ also modulated basal calcium and PoPo3 levels (Supplementary Fig. 3) With TRPA1 inhibition we found that without TNF pre-stimulation, the antagonist A967079 (10 µM) increased intracellular calcium (Fig. ​ (Fig.3m) 3m ) but decreased it when RASF were pre-incubated with TNF (Fig. ​ (Fig.3n). 3n ). A967079 increased PoPo3 uptake under basal conditions (Fig. ​ (Fig.3o) 3o ) but decreased it after TNF pre-incubation (Fig. ​ (Fig.3p). 3p ). Furthermore, A967079 enhanced basal calcium levels and PoPo3 uptake (Supplementary Fig. 3A–D).

Modulation of intracellular calcium levels and PoP3 uptake of RASF after CBD challenge with concomitant inhibition of membrane TRP channels and mitochondrial targets.

Mean intracellular calcium mobilization (a, b, e, f, i, j, m, n, q, r, u, v) and mean PoPo3 uptake (c, d, g, h, k, l, o, p, s, t, w, x) of RASF after CBD challenge and concomitant inhibition with the antagonists given in the figure. n is the number of experiment replicates from 24 patient samples (a, c), 33 patient samples (b, d), and 8 patient samples (n, p). ex (except n, p) n number equals number of replicates and different patient samples. ANOVA with Dunnett’s T3 post-hoc test was used for all comparisons versus (ad). *p < 0.05, ***p < 0.001.

Mitochondrial targets mediate the effects of CBD

We investigated four proposed mitochondrial targets for CBD: the mitochondrial calcium uniporter (MCU), the sodium/calcium exchanger (NCLX) 30 , the voltage-gated anion channel (VDAC1) 8 and the mitochondrial membrane permeability transition pore (mPTP) which initiates apoptotic and necrotic events 31 .While we detected a minor influence of NCLX and MCU (Supplementary results and Supplementary Fig. 2) on intracellular calcium levels and PoPo3 uptake, inhibition of mPTP exerted the strongest influence (Fig. 3q–t ). CBD-induced changes in intracellular calcium were reduced by the mPTP inhibitor CsA (Fig. ​ (Fig.3q). 3q ). In TNF pre-stimulated RASF, CsA reduced calcium levels over all CBD concentrations (Fig. ​ (Fig.3r). 3r ). In addition, CsA accelerated PoPo3 uptake (Fig. 3s, t ) but did not alter basal calcium or PoPo3 levels (Supplementary Fig. 3A–D). Next, we combined CBD with DIDS, which is an inhibitor of VDAC in the outer mitochondrial and the plasma membrane 32,33 . CBD stabilizes a closed conformation of VDAC, which excludes the exchange of metabolites from the cytosol into mitochondria, but enhances its calcium transport function 8,34 . DIDS increased CBD-induced calcium mobilization regardless of TNF pre-stimulation (Fig. 3u, v ) and it completely abolished PoPo3 uptake under all conditions (Fig. 3w, x ). DIDS also increased basal calcium levels in TNF pre-stimulated RASF (Supplementary Fig. 3B) and decreased PoPo3 levels (Supplementary Fig. 3C, D). Since we found TRPA1 to be involved in the effects of CBD (Figs. ​ (Figs.1h 1 h and 3m–p ), we were interested in the cellular localization of this receptor. Since we assumed this to be an intracellular site, we used Thapsigargin to deplete calcium stores in the endoplasmatic reticulum (ER) 35,36 and Gly-Phe-β-naphthylamide (GPN), which disrupts lysosomes and releases calcium stored in this organelle 37,38 . We found that GPN reduced the elevation of intracellular calcium by CBD (p < 0.001, Fig. 4e, f ), while PoPo3 uptake was significantly enhanced (Fig. 4g, h ). Of note, GPN per se induced an increase of intracellular calcium and PoPo3 uptake (Supplementary Fig. 3A–D). Similarly, the inhibitor of the endoplasmic reticulum Ca 2+ -ATPase, Thapsigargin, reduced the CBD-induced increase in intracellular calcium levels (Fig. 4i, j ) and slightly attenuated PoPo3 uptake (Fig. 4k, l ) but increased basal calcium and PoPo3 levels (Supplementary Fig. 3A–D). Cationic and uncharged compounds are taken up by organic cation transporters (OCT) 39,40 and therefore we assessed the effects of Decynium-22 (D22), an inhibitor of all OCT isoforms on intracellular calcium and the uptake of PoPo3. Under all conditions, we found that D22 prevented the increase of intracellular calcium/PoPo3 uptake induced by CBD (Fig. 4m–p ) and it reduced basal intracellular calcium (Supplementary Fig. 3A, B) but slightly elevated PoPo3 levels (Fig. 3c, d ). Lastly, we determined the influence of CBD on the production of IL-6, IL-8, and MMP-3 by RASF. CBD (10 µM and 20 µM) significantly decreased the production of IL-6 (Fig. ​ (Fig.5a), 5a ), which was inhibited by the addition of CsA (Fig. ​ (Fig.5b). 5b ). IL-8 production was modified by CBD (20 µM) alone (Fig. ​ (Fig.5c) 5c ) and the addition of CsA increased IL-8 levels significantly (Fig. ​ (Fig.5d). 5d ). MMP-3 levels were reduced by 20 µM CBD (Fig. ​ (Fig.5e). 5e ). The cytokine-reducing effects of CBD might be related to the reduction in viable cells, since we detected a reduction in cell number at 10 µM and 20 µM CBD which was inhibited by the addition of CsA (Fig. 5g, h ).

Modulation of intracellular calcium levels and PoP3 uptake of RASF by CBD with concomitant manipulation of lysosomal and endoplasmatic reticulum calcium stores or inhibition of organic cation transport.

Mean intracellular calcium mobilization (a, b, e, f, i, j, m, n) and mean PoPo3 uptake (c, d, g, h, k, l, o, p) of RASF after CBD challenge and concomitant inhibition with the antagonists given in the figure. ANOVA with Dunnett’s T3 post-hoc test was used for all comparisons. ***p < 0.001.

Discussion

In this study, we demonstrated that CBD decreases cell viability, proliferation, and cytokine production but increases intracellular calcium and PoPo3 levels of RASF and all effects were enhanced by TNF pre-stimulation. These effects were mediated by TRPA1 and by the assembly of the mPTP under pro-inflammatory conditions, whereas under unstimulated conditions, TRPA1 was not involved.

We demonstrated that CBD reduces cell viability, but RealTime-Glo assays were conducted in serum-free medium without carrier protein. Therefore, we assessed whether CBD influences RASF proliferation in medium containing FCS, since in vivo, CBD is bound to serum albumin, which lowers its free concentration available for receptor binding 24,41 . We confirmed that the anti-proliferative effect of CBD is dependent on FCS content. Consequently, for in vivo applications, CBD needs to be administered in high concentrations to elicit beneficial effects as shown in the treatment of Dravet syndrome 13 . In order to identify the cellular targets for CBD, we used the TRPA1 antagonist A967079 and the pan-TRP antagonist RR to inhibit the effects of CBD on cell viability as CBD has already been identified as ligand for TRPA1 23 . Moreover, we found that CsA reversed the detrimental effects of CBD on cell viability, which confirms results from Olivas-Aguirre et al. 25 that showed mitochondrial calcium overload correlates with assembly of the mPTP by CBD. It has been demonstrated that CBD influences calcium homeostasis 25 and we also found CBD to elevate intracellular calcium in RASF. This confirms own previous results demonstrating calcium mobilization in response to TRPA1 ligation 22 . Moreover, we showed that TNF up-regulates TRPA1 protein in RASF, which translates into increased sensitivity to TRPA1 ligands 22 . CBD also increased calcium levels without extracellular calcium by using PBS instead of HBSS, suggesting mobilization form intracellular stores. In fact, in dorsal root ganglia neurons it has been shown that TRPA1 is located in lysosomes, where its activation fosters neurotransmitter release 42 . Although we do not provide direct evidence regarding the localization of TRPA1, the use of the cell-impermeable pan-TRP inhibitor RR 43 , which was only able to slightly attenuate the effects of CBD suggests an intracellular target protein. In line with this, the lipophilic antagonist A967079 decreased calcium mobilization and PoPo3 uptake after CBD challenge. Moreover, we used Thapsigargin to deplete ER calcium stores and GPN to disrupt lysosomes and both compounds reduced the elevation of intracellular calcium after CBD exposure. This shows that ER calcium stores are involved and it has been shown that even if calcium originates from lysosomes, the signal is amplified by depletion of ER stores 44 . This is important, because although GPN has been reported to mobilize lysosomal calcium, a recent study claimed that GPN increases calcium through an ER-dependent mechanism 45 . CBD is also an agonist at TRPV1/2 ion channels 7,23,46,47 , but neither CPZ nor RR inhibited the effects of CBD, ruling out these receptors as target molecules. CPZ is also an agonist at TRPA1 48 , and we did detect a small increase in basal intracellular calcium in response to this ligand alone. Accordingly, CPZ slightly elevated the calcium response of RASF to CBD suggesting a sensitizing effect on TRPA1. TRPA1 inhibition with A967079 reduced calcium mobilization and PoPo3 uptake in TNF pre-stimulated but not naïve RASF, where we found the opposite, suggesting that CBD exerts additional effects via different cellular targets besides TRPA1. This demonstrates that TRPA1 contributes to the rise in intracellular calcium only in TNF pre-stimulated RASF, in which TRPA1 is upregulated. Besides binding to TRPs, it has been demonstrated that CBD ligates several proteins 11,49 with mitochondrial targets being the most prominent 8–10,25,30 . In mitochondria, CBD targets VDAC1, NCLX, MCU, and controls assembly of the mPTP 8–10,25 . Although protective effects of CBD against mitochondrial toxins have been shown 10,50 , the majority of studies demonstrated that CBD induces cell death by disturbing calcium homeostasis 8,9,25 . This confirms our results, since CBD augmented calcium levels with a concomitant increase in cell death. Excess cytosolic calcium is taken up by mitochondria which are depolarized in this process 51 . If mitochondrial calcium levels exceed a certain threshold, mPTP is assembled leading to cell death 52 . In fact, we demonstrated that only CsA prevented cell death, suggesting that mitochondrial calcium overload occurs in CBD-stimulated RASF. In line with this, CsA reduced intracellular calcium, which is another indicator that mitochondria provide a significant contribution to the increase in calcium by CBD. Calcium is increased by mPTP formation as mitochondria are permeabilized releasing stored calcium into the cytosol 53 . Thus, inhibiting mPTP formation by CsA decreases calcium leakage from mitochondria and subsequent cell death. In addition, we found that the NCLX inhibitor CGP reduced cytosolic calcium levels. CGP blocks calcium transport from the mitochondrial matrix into the intermembrane space, thus increasing mitochondrial and reducing cytosolic calcium levels 54 . We also used the reverse mode inhibitor of the NCLX, KB-R7943, but results with this inhibitor are difficult to interpret due to its interaction with the MCU and NCLX. In the latter it can act as forward or reverse mode antagonist dependent on cell type, NCLX isoform, and concentration 55,56 . Using the specific MCU inhibitor DS16570511 we found increased cytosolic calcium levels in TNF pre-stimulated but not in unstimulated RASF. This might be explained by TRPA1, which only contributed to calcium level alterations in TNF pre-treated RASF. For the inhibition of VDAC1 we used DIDS which increased intracellular calcium in unstimulated, but decreased calcium in TNF pre-treated RASF. This might depend on the initial calcium signal generated by CBD, because DIDS can permeabilize the inner mitochondrial membrane depending on calcium concentration leading to formation of mPTP 57 . Besides calcium, PoPo3 uptake served as readout for changes in cell viability as it is supposed to enter cells with a compromised plasma membrane only. However, several studies showed that the uptake of PoPo3 related compounds also occurs via specific receptors/ion channels 58,59 . In addition, it has been demonstrated that the family of organic cation transporters (OCT) mediates the uptake of many charged but also electroneutral compounds into the cell 39 . Therefore, it is quite possible that PoPo3 is also taken up by OCT and indeed we show that decynium-22, which inhibits all OCT isoforms 39 strongly reduced PoPo3 uptake and it also blunted the increase of intracellular calcium, which might be due to the electrogenic properties of D22 40 . Another possibility is that OCT mediates the uptake of CBD and D22 would limit the access of CBD to intracellular compartments. D22 did not influence basal uptake of PoPo3, but reduced the CBD-induced uptake and this might be related to changes in intracellular calcium since the activity of OCT is regulated by calcium-dependent proteins 39 . DIDS completely blocked PoPo3 uptake but these results are difficult to interpret since DIDS does not inhibit OCT but membrane anion channels, which should not mediate the uptake of the cationic dye PoPo3. It might be that the negatively charged DIDS binds PoPo3 directly, thereby inhibiting binding to DNA and the increase in fluorescence. PoPo3 might be suitable as a surrogate marker for the uptake of chemical compounds/drugs, which is enhanced by CBD. Since RASF produce high amounts of IL-6, IL-8, and MMP-3 60 , we also investigated the impact of CBD on production of these mediators. CBD dose-dependently reduced IL-6, IL-8, and MMP-3 with concomitant reduction in cell viability. CsA was able to rescue RASF from cell death and increased Il-6 and IL-8 production confirming that cell death is the influencing factor on cytokine production.

From our data, we propose a mechanism of how CBD influences RASF function and induces cell death under pro-inflammatory conditions (Fig. ​ (Fig.6). 6 ). TNF sensitizes RASF to the action of CBD by up-regulating TRPA1 22 . CBD increases intracellular calcium by activating TRPA1 but it also binds several mitochondrial targets like VDAC1, MCU, and NCLX, which on their part influence cytosolic calcium. Eventually, mitochondrial calcium overload occurs and mPTP is assembled leading to cell death.

TNF increases TRPA1 protein, which is located in intracellular compartments. CBD activates TRPA1 and Ca 2+ is released into the cytosol. Elevations in cytosolic Ca 2+ are reduced through uptake into mitochondria. In addition, increased cytosolic Ca 2+ might enhance the activity of organic cation transporters (OCT) which might mediate the uptake of the fluorescent dye PoPo3. Additionally, OCT might mediate the uptake of CBD itself. By binding to VDAC1, CBD increases Ca 2+ flux through the outer mitochondrial membrane. Ca 2+ is then taken up into the matrix by the mitochondrial Ca 2+ uniporter (MCU) and, if mitochondria are depolarized, by the Na + /Ca 2+ exchanger (NCLX), which operates in reverse mode under these conditions. Ca 2+ overload occurs, the mitochondrial permeability transition pore (mPTP) assembles and cell death occurs.

CBD has been used in animal model of RA, demonstrating anti-inflammatory and analgesic effects but the mechanism of action has not been identified 17,19 . Here, we demonstrate that CBD reduces cell viability preferentially in TNF-activated RASF via TRPA1 and mitochondrial targets. CBD might decrease chronic inflammation since RA is characterized by a hypoxic environment in the joint with concomitant mitochondrial dysfunction 61 . In this setting, immune cells and RASF might be specifically vulnerable to a “second hit” induced by CBD, leading to deletion of pro-inflammatory immune cells and fibroblasts thereby resolving inflammation. In addition, CBD might also synergize with anti-rheumatic drugs like methotrexate or JAK inhibitors, since it has been reported in tumor cell lines that CBD works in synergy with e.g. the chemotherapeutic drug doxorubicin 62 . Furthermore, CBD also targets TRPV2, which not only increases the uptake of cytotoxic chemotherapeutic agents but also reduces RASF invasion and matrix metalloproteinase production 47,63 . In conclusion, CBD might be beneficial as an adjuvant treatment in rheumatoid arthritis that might support the action of currently used disease-modifying anti-rheumatic drugs.

Materials and methods

Biochemicals

Patients

In total, 40 patients with long-standing RA fulfilling the American College of Rheumatology revised criteria for RA (24) were included in this study. The RA group comprised of 32 females and 8 males with a mean age of 67.8 years ±10.5 years and 66.9 years ±8.2 years, respectively. C-reactive protein was 47.9 mg/dl ± 186.3 mg/dL for females and 28.7 mg/dL ± 43.2 mg/dL for males and rheumatoid factor was 184.4 iU/mL ± 280.4 iU/mL for females and 31.8 iU/mL ± 37.6 iU/mL for males. IN all, 11 out of 40 patients received glucocorticoids, 7 out of 40 methotrexate, 3 out of 40 biologicals, and 1 out of 40 a JAK inhibitor. All patients underwent elective knee joint replacement surgery, and they were informed about the purpose of the study and gave written consent. The study was approved by the Ethics Committees of the University of Düsseldorf (approval number 2018-87-KFogU) and Regensburg (approval number 15-1 01-021). We confirm that all experiments were performed in accordance with relevant guidelines and regulations (Table ​ (Table1 1 ).

Table 1

Biochemicals used in this study.

order # vendor solvent [c] used in experiments Reference
DIDS 4523 Tocris DMSO 50 µM 32,64
Cannabidiol 1570 Tocris DMSO 0.5 µM, 1 µM, 5 µM, 10 µm, 20 µM 7,8,23
> CGP37157 1114 Tocris DMSO 1 µM, 10 µM 30,65
KB-R7943 1244 Tocris DMSO 2.5 µM, 25 µM 55,56
A967079 4716 Tocris DMSO 10 µM 22,66,67
Ruthenium red 1439 Tocris H2O 10 µM 68,69
Cyclosporin A 30024 Sigma DMSO 10 µM 9
Capsazepine 464 Tocris DMSO 10 µM 29,70
GPN 14634 Cayman DMSO 250 µM 29,70,71
Thapsigargin 1138/1 Tocris DMSO 10 µM 72
Decynium-22 4722 Tocris DMSO 10 µM 39,40

The concentrations used in this study are based on values found in the literature (reference).

Synovial fibroblast and tissue preparation

Samples from RA synovial tissue were isolated and prepared as described previously 22 (for details see also Supplementary methods).

Proliferation of RASF

Proliferation was assessed by the cell titer blue viability assay (Promega, Madison, USA, # G8080) according to manufacturer’s instructions.

Intracellular calcium and PoPo3 uptake

In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, abcam, Cambridge, UK) in Hanks buffered salt solution (1 mM Ca 2+ ; HBSS, sigma, # 55037 C) or PBS (no Ca 2+ ) with 0.02% Pluoronic F127 (Thermo fisher scientific, Waltham, USA, # P6866) for 60 min at 37 °C followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, CBD was added and the intracellular Ca 2+ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.

Flow cytometry

RASF were trypsinized, washed and fixed for 20 min with 3.7% formaldehyde (F8775, Sigma Aldrich). Cells were permeabilized with 0.1% Triton X-100 (X100, Sigma) in PBS for 10 min. Then, 0.2 µg/50 µl primary antibody (Proteintech, 19124-1-AP) was added for 2 h. The secondary antibody (Abcam, goat anti-rabbit IgG H&L (Alexa Fluor® 488), ab150077) was incubated for 1 h. Cells were analyzed using a MACS Quant 9 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).

RealTime-Glo cell viability assay

Cell viability was assessed according to manufacturer’s instructions (Promega, # G9711).

Statistical analysis

Statistical analysis was performed with SPSS 25 (IBM, Armonk, USA). The statistic tests used are given in the figure legends. Normal distribution was determined using the Shapiro–Wilk test, equal variance was determined by Levene’s test. In the case of equal variance, the Bonferroni post-hoc test was used, otherwise the Dunnet’s post-hoc test was employed. When data are presented as box plots, the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. When data are presented as line plots, the line represents the mean. When data are presented as bar charts, the top of the bar represents the mean and error bars depict the standard error of the mean (sem). The level of significance was p < 0.05.

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